Methods of making l-ornithine phenyl acetate

ABSTRACT

Disclosed herein are processes for making L-ornithine phenyl acetate. The process may include, for example, intermixing a halide salt of L-ornithine with silver phenyl acetate. The process may also include forming a phenyl acetate salt in situ. The present application also relates to various compositions obtained from these processes, including crystalline forms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/878,146, filed Apr. 5, 2013, which is the U.S. National Phase under35 U.S.C. §371 of International Application No. PCT/US2011/054983,entitled “METHODS OF MAKING L-ORNITHINE PHENYL ACETATE,” filed Oct. 5,2011, and published in English on Apr. 12, 2012 as WO 2012/048043, whichclaims the benefit of priority to U.S. Provisional Application No.61/390,585, filed Oct. 6, 2010, each of which is hereby incorporated byreference in its entirety. Any and all applications for which a foreignor domestic priority claim is identified in the Application Data Sheetas filed with the present application, or any correction thereto, arehereby incorporated by reference under 37 CFR 1.57.

BACKGROUND

1. Field

The present application relates to the fields of pharmaceuticalchemistry, biochemistry, and medicine. In particular, it relates tomethods of making L-ornithine phenyl acetate salts.

2. Description

Hyperammonemia is a hallmark of liver disease and is characterized by anexcess of ammonia in the bloodstream. Hepatic encephalopathy is aprimary clinical consequence of progressive hyperammonemia and is acomplex neuropsychiatric syndrome, which may complicate acute or chronichepatic failure. It is characterized by changes in mental stateincluding a wide range of neuropsychiatric symptoms ranging from minorsigns of altered brain function to overt psychiatric and/or neurologicalsymptoms, or even deep coma. The accumulation of unmetabolized ammoniahas been considered as the main factor involved in the pathogenesis ofhepatic encephalopathy, but additional mechanisms may be associated.

L-Ornithine monohydrochloride and other L-ornithine salts are availablefor their use in the treatment of hyperammonemia and hepaticencephalopathy. For example, U.S. Publication No. 2008/0119554, which ishereby incorporated by reference in its entirety, describes compositionsof L-ornithine and phenyl acetate for the treatment of hepaticencephalopathy. L-ornithine has been prepared by enzymatic conversionmethods. For example, U.S. Pat. Nos. 5,405,761 and 5,591,613, both ofwhich are hereby incorporated by reference in their entirety, describeenzymatic conversion of arginine to form L-ornithine salts. Sodiumphenyl acetate is commercially available, and also available as aninjectable solution for the treatment of acute hyperammonemia. Theinjectable solution is marketed as AMMONUL.

Although salt forms may exhibit improved degradation properties, certainsalts, particularly sodium or chloride salts, may be undesirable whentreating patients having diseases associated with the liver disease,such as hepatic encephalopathy. For example, a high sodium intake may bedangerous for cirrhotic patients prone to ascites, fluid overload andelectrolyte imbalances. Similarly, certain salts are difficult toadminister intravenously because of an increased osmotic pressure, i.e.,the solution is hypertonic. High concentrations of excess salt mayrequire diluting large volumes of solution for intravenousadministration which, in turn, leads to excessive fluid overload.Accordingly, there exists a need for the preparation of L-ornithine andphenyl acetate salts which are favorable for the treatment of hepaticencephalopathy or other conditions where fluid overload and electrolyteimbalance are prevalent.

SUMMARY

Some embodiments disclosed herein include a process for makingL-ornithine phenyl acetate.

Some embodiments disclosed herein include a process for makingL-ornithine phenyl acetate salt comprising intermixing an L-ornithine,or a salt thereof, and phenyl acetic acid, or a salt thereof.

Some embodiments include a process for making L-ornithine phenyl acetatesalt comprising intermixing a phenyl acetate salt, L-ornithine benzoate,and a solvent; and isolating a composition comprising at least 70%crystalline L-ornithine phenyl acetate by weight.

In some embodiments, the process further includes forming L-ornithinebenzoate by intermixing an L-ornithine salt, a benzoate salt and a firstsolvent to form an intermediate solution.

In some embodiments, the process further includes removing at least aportion of a salt from said intermediate solution before intermixing thephenyl acetate salt, wherein said salt is not an L-ornithine salt.

In some embodiments, the salt removed from the intermediate solutioncomprises an anion derived at least in part from the L-ornithine saltand a cation derived at least in part from the benzoate salt.

In some embodiments, the L-ornithine salt is L-ornithine hydrochlorideand said anion is chloride.

In some embodiments, the benzoate salt is silver benzoate and the cationis a silver ion.

In some embodiments, the process further comprises adding hydrochloricacid before removing at least a portion of the salt.

In some embodiments, at least about 90% by weight of the salt is removedfrom the intermediate solution.

In some embodiments, the process further comprises forming L-ornithinebenzoate by intermixing an L-ornithine salt, a benzoate salt and asolvent to form an intermediate solution, and isolating L-ornithinebenzoate from said intermediate solution.

In some embodiments, the process further comprises removing at least aportion of a salt from the intermediate solution before isolating theL-ornithine benzoate, wherein the salt is not an L-ornithine salt.

In some embodiments, the process further comprises adding hydrochloricacid before removing at least a portion of the salt.

In some embodiments, the isolation of L-ornithine benzoate comprisescrystallizing L-ornithine benzoate from the intermediate solution.

In some embodiments, the phenyl acetate salt is dispersed in a solutionwhich is intermixed with L-ornithine benzoate and the solvent.

Some embodiments include a process for making L-ornithine phenyl acetatesalt comprising: preparing a solution of phenyl acetate salt by mixing aphenyl acetic acid and an appropriate base in a first solvent;intermixing an L-ornithine benzoate with the solution of phenyl acetatesalt; and isolating a composition comprising L-ornithine phenyl acetate.

In some embodiments, the appropriate base is selected from the groupconsisting of an alkali metal hydroxide and an alkali metal alkoxide.

In some embodiments, the process further comprises forming L-ornithinebenzoate by intermixing an L-ornithine salt, a benzoate salt and asecond solvent to form an intermediate solution.

In some embodiments, the composition comprises at least about 0.10% byweight benzoate salt.

In some embodiments, the composition comprises no more than 5% by weightbenzoate salt.

In some embodiments, the composition comprises no more than 3% by weightbenzoate salt.

In some embodiments, the composition comprises no more than 1% by weightbenzoate salt.

In some embodiments, the L-ornithine salt is L-ornithine hydrochloride.

In some embodiments, the benzoate salt is silver benzoate.

In some embodiments, the composition further comprises at least 10 ppmsilver.

In some embodiments, the composition comprises at least 20 ppm silver.

In some embodiments, the composition comprises at least 25 ppm silver.

In some embodiments, the composition comprises no more than 600 ppmsilver.

In some embodiments, the composition comprises no more than 100 ppmsilver.

In some embodiments, the composition comprises no more than 65 ppmsilver.

In some embodiments, the phenyl acetate is an alkali metal salt.

In some embodiments, the alkali metal salt is sodium phenyl acetate.

In some embodiments, the composition comprises no more than 100 ppmsodium.

In some embodiments, the composition comprises no more than 20 ppmsodium.

In some embodiments, the L-ornithine salt is a halide salt.

In some embodiments, the L-ornithine halide salt is L-ornithinehydrochloride.

In some embodiments, the composition comprises no more than 0.1% byweight chloride.

In some embodiments, the composition comprises no more than 0.01% byweight chloride.

Some embodiments include a process for making L-ornithine phenyl acetatesalt comprising: increasing the pH value of a mixture comprising anL-ornithine salt at least until an intermediate salt precipitates,wherein said intermediate salt is not an L-ornithine salt; isolating theintermediate salt from said mixture; intermixing phenyl acetic acid withsaid mixture; and isolating L-ornithine phenyl acetate salt from saidsolution.

In some embodiments, the pH value is increased to at least 8.0.

In some embodiments, the pH value is increased to at least 9.0.

In some embodiments, increasing the pH value comprises adding a pHmodifier selected from the group consisting of sodium hydroxide,potassium hydroxide, cesium hydroxide, lithium hydroxide, calciumhydroxide, magnesium hydroxide, barium hydroxide, ammonium hydroxide,sodium carbonate, sodium bicarbonate, potassium carbonate, potassiumbicarbonate, calcium carbonate, magnesium carbonate, barium carbonate,sodium methoxide, potassium t-butoxide, dibutylamine, tryptamine,lithium hydride, sodium hydride, calcium hydride, butyl lithium, ethylmagnesium bromide or combinations thereof.

In some embodiments, the intermediate salt comprises an anion derived atleast in part from the L-ornithine salt.

In some embodiments, the intermediate salt comprises a cation derived atleast in part from the pH modifier.

In some embodiments, the pH modifier is selected from the groupconsisting of sodium hydroxide, sodium methoxide, calcium hydroxide,calcium carbonate and barium hydroxide.

Some embodiments include a process for making L-ornithine phenyl acetatesalt comprising intermixing an L-ornithine salt, a phenyl acetate saltand a solvent to form a solution, and isolating L-ornithine phenylacetate from said solution.

In some embodiments, the L-ornithine salt is a halide salt. In someembodiments, the halide salt is not L-ornithine hydrochloride.

In some embodiments, the phenyl acetate salt is silver phenyl acetate.

In some embodiments, the L-ornithine salt is L-ornithine hydrochloride.

Some embodiments include compositions of L-ornithine phenyl acetateprepared according to the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction pattern of Form I.

FIG. 2 shows differential scanning calorimetry results for Form I.

FIG. 3 shows thermogravimetric gravimetric/differential thermal analysisof Form I.

FIG. 4 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form I.

FIG. 5 shows dynamic vapor sorption results for Form I.

FIG. 6 is an X-ray powder diffraction pattern of Form II.

FIG. 7 shows differential scanning calorimetry results for Form II.

FIG. 8 shows thermogravimetric gravimetric/differential thermal analysisof Form II.

FIG. 9 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form II.

FIG. 10 shows dynamic vapor sorption results for Form II.

FIG. 11 is an X-ray powder diffraction pattern of Form III.

FIG. 12 shows differential scanning calorimetry results for Form III.

FIG. 13 shows thermogravimetric gravimetric/differential thermalanalysis of Form III.

FIG. 14 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form III.

FIG. 15 shows dynamic vapor sorption results for Form III.

FIG. 16 is an X-ray powder diffraction pattern of Form V.

FIG. 17 shows differential scanning calorimetry results for Form V.

FIG. 18 shows thermogravimetric gravimetric/differential thermalanalysis of Form V.

FIG. 19 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form V.

FIG. 20 shows dynamic vapor sorption results for Form V.

FIG. 21 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of L-ornithine benzoate.

FIG. 22 shows the 1H nuclear magnetic resonance spectrum obtained from asample of L-ornithine phenyl acetate.

DETAILED DESCRIPTION

Some embodiments disclosed herein include a method of making L-ornithinephenyl acetate salt. L-Ornithine phenyl acetate may be produced, forexample, through an intermediate salt, such as L-ornithine benzoate. Asshown in Scheme 1, an L-ornithine salt of Formula I can be reacted witha benzoate salt of Formula II to obtain the intermediate L-ornithinebenzoate.

Various salts of L-ornithine may be used in the compound of Formula I,and therefore X in Formula I can be any ion capable of forming a saltwith L-ornithine other than benzoic acid or phenyl acetic acid. X can bea monoatomic anion, such as, but not limited to, a halide (e.g.,fluoride, chloride, bromide, and iodide). X can also be a polyatomicanion, such as, but not limited to, acetate, aspartate, formate,oxalate, bicarbonate, carbonate, sulfate, nitrate, isonicotinate,salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, glutamate, methanesulfonate, ethanesulfonate,benzensulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate), phosphate and the like. Xmay be an organic or inorganic group. In some embodiments, X is amonovalent ion. In some embodiments, X is chloride.

Similarly, the benzoate salt of Formula II is not particularly limited,and therefore Y in Formula II can be any appropriate ion capable offorming a salt with benzoic acid. In some embodiments, Y can be amonoatomic cation, such as an alkali metal ion (e.g., Li⁺, Na⁺, and K⁺)and other monovalent ions (e.g., A_(g) ⁺). Y may also be a polyatomiccation, such as ammonium, L-arginine, diethylamine, choline,ethanolamine, 1H-imidazole, trolamine, and the like. In someembodiments, Y is an inorganic ion. In some embodiments, Y is silver.

Many other possible salts of L-ornithine and benzoic acid may be usedfor the compounds of Formulae I and II, respectively, and can readily beprepared by those skilled in the art. See, for example, Bighley L. D.,et al., “Salt forms of drugs and absorption,” In: Swarbrick J., HorlanJ. C., eds. Encyclopedia of pharmaceutical technology, Vol. 12. NewYork: Marcel Dekker, Inc. pp. 452-499, which is hereby incorporated byreference in its entirety.

The intermediate L-ornithine benzoate (i.e., Formula III) can beprepared by intermixing solutions including compounds of Formulae I andII. As an example, the compounds of Formulae I and II may be separatelydissolved in water and dimethyl sulfoxide (DMSO), respectively. The twosolutions may then be intermixed so that the L-ornithine and benzoicacid react to form the salt of Formula III. Alternatively, the two saltcompounds can be directly dissolved into a single solution. In someembodiments, L-ornithine and benzoic acid are dissolved in separatesolvents, and subsequently intermixed. In some embodiments, L-ornithineis dissolved in an aqueous solution, benzoic acid is dissolved in anorganic solvent, and the L-ornithine and benzoic acid solutions aresubsequently intermixed.

Non-limiting examples of solvents which may be used when intermixingL-ornithine and benzoate salts include acetonitrile, dimethylsulfoxide(DMSO), cyclohexane, ethanol, acetone, acetic acid, 1-propanol,dimethylcarbonate, N-methyl-2-pyrrolidone (NMP), ethyl acetate (EtOAc),toluene, isopropyl alcohol (IPA), diisopropoyl ether, nitromethane,water, 1,4-dioxane, diethyl ether, ethylene glycol, methyl acetate(MeOAc), methanol, 2-butanol, cumene, ethyl formate, isobutyl acetate,3-methyl-1-butanol, anisole, and combinations thereof. In someembodiments, the L-ornithine benzoate solution includes water. In someembodiments, the L-ornithine benzoate solution includes DMSO.

Upon intermixing L-ornithine and benzoate salts, counterions X and Y mayform a precipitate that can be removed from the intermixed solutionusing known methods, such as filtration, centrifugation, and the like.In some embodiments, X is chloride, Y is silver, and the reactionproduces a precipitate having AgCl. Although Scheme 1 shows thecompounds of Formulae I and II as salts, it is also within the scope ofthe present application to intermix the free base of L-ornithine andbenzoic acid to form the intermediate of L-ornithine benzoate.Consequently, forming and isolating the precipitate is optional.

The relative amount of L-ornithine and benzoate salts that areintermixed is not limited; however the molar ratio of L-ornithine tobenzoic acid may optionally be in the range of about 10:90 and 90:10. Insome embodiments, the molar ratio of L-ornithine benzoate can be in therange of about 30:70 and 70:30. In some embodiments, the molar ratio ofL-ornithine to benzoate can be in the range of about 40:60 and 60:40. Insome embodiments, the molar ratio of L-ornithine to benzoate is about1:1.

In embodiments where X and Y are both inorganic ions (e.g., X and Y arechloride and silver, respectively), additional amounts of X-containingsalt may be added to encourage further precipitation of the counterionY. For example, if X is chloride and Y is silver, the molar ratio ofL-ornithine hydrochloride to silver benzoate may be greater than 1:1 sothat an excess of chloride is present relative to silver. Accordingly,in some embodiments, the molar ratio of L-ornithine to benzoic acid isgreater than about 1:1. Nevertheless, the additional chloride salt isnot required to be derived from an L-ornithine salt (e.g., L-ornithinehydrochloride). For example, dilute solutions of hydrochloric acid maybe added to the solution to further remove silver. Although it is notparticularly limited when the additional X-containing salt is added, itis preferably added before the AgCl is initially isolated.

As shown in Scheme 2, the L-ornithine benzoate can react with a phenylacetate salt of Formula IV to form L-ornithine phenyl acetate. Forexample, sodium phenyl acetate can be intermixed with a solution ofL-ornithine benzoate to form L-ornithine phenyl acetate. Various saltsof phenyl acetate may be used, and therefore Z in Formula IV can be anycation capable of forming a salt with phenyl acetate other than benzoicacid or L-ornithine. In some embodiments, Z can be a monoatomic cation,such as an alkali metal ion (e.g., Li⁺, Na⁺, and K⁺) and othermonovalent ions (e.g., Ag⁺). Z may also be a polyatomic cation, such asammonium, L-arginine, diethylamine, choline, ethanolamine, 1H-imidazole,trolamine, and the like. In some embodiments, Z is an inorganic ion. Insome embodiments, Z is sodium.

The phenyl acetate salt may optionally be prepared in solution usingphenyl acetic acid and an appropriate base. This solution may beintermixed with L-ornithine benzoate to obtain L-ornithine phenylacetate as described above. As an example, phenyl acetic acid may beintermixed with sodium hydroxide in isopropanol to obtain a solution ofsodium phenyl acetate. The solution of sodium phenyl acetate can then beintermixed with a solution of L-ornithine benzoate. Alternatively, thephenyl acetate salt may optionally be isolated as a solid beforeintermixing with L-ornithine benzoate.

The base for preparing phenyl acetate salt is not particularly limitedand will be selected, in part, based upon the desired phenyl acetatesalt. As an example, sodium phenyl acetate may be obtained by addingsodium hydroxide or sodium methoxide. The base can be an inorganic baseor an organic base. In some embodiments, the base is an alkali metalbase. For example, the base may include lithium hydroxide, sodiumhydroxide, and potassium hydroxide. In some embodiments, the base is analkaline earth metal salt. As an example, the base may include calciumhydroxide, magnesium hydroxide, and barium hydroxide. In someembodiments, the base is water-soluble. Non-limiting examples of basesinclude sodium hydroxide, potassium hydroxide, calcium hydroxide,magnesium hydroxide, sodium methoxide, potassium methoxide, calciummethoxide, magnesium methoxide, sodium tert-butoxide, potassiumtert-butoxide, calcium tert-butoxide, and magnesium tert-butoxide

The relative amount of L-ornithine salt and phenyl acetate salt that areintermixed is also not limited; however the molar ratio of L-ornithineto phenyl acetate may optionally be in the range of about 10:90 and90:10. In some embodiments, the molar ratio of L-ornithine to phenylacetate can be in the range of about 30:70 and 70:30. In someembodiments, the molar ratio of L-ornithine to phenyl acetate can be inthe range of about 40:60 and 60:40. In some embodiments, the molar ratioof L-ornithine to phenyl acetate is about 1:1.

The L-ornithine phenyl acetate of Formula V may then be isolated fromsolution using known techniques. For example, by evaporating any solventuntil the L-ornithine phenyl acetate crystallizes, or alternatively bythe adding an anti-solvent miscible in the L-ornithine phenyl acetatesolution until the L-ornithine phenyl acetate precipitates fromsolution. Another possible means for isolating the L-ornithine phenylacetate is to adjust the temperature of the solution (e.g., lower thetemperature) until the L-ornithine phenyl acetate precipitates.

The method of isolating the L-ornithine phenyl acetate affects thecrystalline form that is obtained. The crystalline forms are discussedfurther below and are also disclosed in three related applications: (i)U.S. Provisional Application No. 61/166,676, filed Apr. 3, 2009; (ii)PCT/US2010/029708, filed in English on Apr. 1, 2010; and (iii) U.S.application Ser. No. 12/753,763, filed Apr. 2, 2010. These applicationsare hereby incorporated by reference in their entirety.

The isolated L-ornithine phenyl acetate may be subjected to variousadditional processing, such as drying and the like. In some embodiments,L-ornithine phenyl acetate may be subsequently intermixed with a diluteHCl solution to precipitate residual silver. The L-ornithine phenylacetate may again be isolated from solution using similar methodsdisclosed above.

As would be appreciated by a person of ordinary skill in the art, guidedby the teachings of the present application, L-ornithine phenyl acetatemay similarly be prepared using an intermediate salt other thanL-ornithine benzoate. Thus, for example, L-ornithine, or a salt thereof(e.g., L-ornithine hydrochloride), can be intermixed with a solutionhaving acetic acid. L-Ornithine acetate may then be intermixed withphenyl acetic acid, or a salt thereof (e.g., sodium phenyl acetate), toobtain L-ornithine phenyl acetate. Scheme 3 illustrates one example of aprocess for forming L-ornithine phenyl acetate using L-ornithine acetateas an intermediate salt.

Other salts may be used besides benzoate and acetate. In someembodiments, the intermediate salt can be a pharmaceutically acceptablesalt of L-ornithine. For example, the intermediate L-ornithine salt canbe an acetate, aspartate, formate, oxalate, bicarbonate, carbonate,sulfate, nitrate, isonicotinate, salicylate, citrate, tartrate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucaronate, saccharate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate,pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate) or phosphate.The free acid of the intermediate is preferably a weaker acid relativeto phenyl acetic acid. In some embodiments, the intermediate is anL-ornithine salt with an anion component that exhibits a pK_(a) valuethat is higher than the pK_(a) value of phenyl acetic acid. As anexample, for L-ornithine acetate, acetic acid and phenyl acetic acidexhibit pK_(a) values of about 4.76 and 4.28, respectively.

L-Ornithine phenyl acetate may also be prepared, in some embodiments,without forming an intermediate salt, such as L-ornithine benzoate.Scheme 4 illustrates an exemplary process for preparing L-ornithinephenyl acetate without an intermediate salt. A pH modifier may be addedto a solution of L-ornithine salt (e.g., as illustrated in Scheme 4 bythe compound of Formula I) until a salt precipitates from solution,where the salt is not an L-ornithine salt. As an example, sodiummethoxide (NaOMe) can be added to a mixture of L-ornithine hydrochlorideuntil sodium chloride precipitates from solution to leave a free base ofL-ornithine. The precipitate may optionally be isolated from solutionusing known techniques, such as filtration, centrifugation, and thelike. The free base of L-ornithine (e.g., as illustrated in Scheme 4 bythe compound of Formula I-a) may be intermixed with phenyl acetic acid,or a salt thereof (e.g., as illustrated in Scheme 4 by the compound ofFormula IV), to obtain L-ornithine phenyl acetate. The L-ornithinephenyl acetate of Formula V may then be isolated as previouslydescribed.

A pH modifier can include a basic compound, or anhydrous precursorthereof, and/or a chemically protected base. Non-limiting examples of pHmodifiers include sodium hydroxide, potassium hydroxide, cesiumhydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide,barium hydroxide, ammonium hydroxide, sodium carbonate, sodiumbicarbonate, potassium carbonate, potassium bicarbonate, calciumcarbonate, magnesium carbonate, barium carbonate, sodium methoxide,potassium methoxide, sodium t-butoxide, potassium t-butoxide,dibutylamine, tryptamine, lithium hydride, sodium hydride, calciumhydride, butyl lithium, ethyl magnesium bromide and combinationsthereof. Also, the amount of pH modifier to be added is not particularlylimited; however the molar ratio of L-ornithine to pH modifier mayoptionally be in the range of about 10:90 and 90:10. In someembodiments, the molar ratio of L-ornithine to pH modifier can be in therange of about 30:70 and 70:30. In some embodiments, the molar ratio ofL-ornithine to pH modifier can be in the range of about 40:60 and 60:40.In some embodiments, the molar ratio of L-ornithine to pH modifier isabout 1:1. The pH modifier may, in some embodiments be added to adjustthe pH value to at least about 8.0; at least about 9.0; or at leastabout 9.5.

Another process for forming L-ornithine phenyl acetate, in someembodiments, includes reacting a halide salt of L-ornithine with silverphenyl acetate (Scheme 5). As an example, L-ornithine hydrochloride maybe intermixed with silver phenyl acetate and a solvent. AgCl may thenprecipitate and is optionally isolated from the solution. The remainingL-ornithine phenyl acetate can also be isolated using known methods.This process can be completed using generally the same procedures andconditions outlined above.

The relative amount of L-ornithine salt and phenyl acetate salt that areintermixed is also not limited; however the molar ratio of L-ornithineto phenyl acetate may optionally be in the range of about 10:90 and90:10. In some embodiments, the molar ratio of L-ornithine to phenylacetate can be in the range of about 30:70 and 70:30. In someembodiments, the molar ratio of L-ornithine to phenyl acetate can be inthe range of about 40:60 and 60:40. In some embodiments, the molar ratioof L-ornithine to phenyl acetate is about 1:1.

The L-ornithine phenyl acetate may then be isolated from solution usingknown techniques. For example, by evaporating any solvent until theL-ornithine phenyl acetate crystallizes, or alternatively by the addingan anti-solvent miscible in the L-ornithine phenyl acetate solutionuntil the L-ornithine phenyl acetate precipitates from solution. Anotherpossible means for isolating the L-ornithine phenyl acetate is to adjustthe temperature of the solution (e.g., lower the temperature) until theL-ornithine phenyl acetate precipitates.

The compositions obtained according to the methods disclosed herein canbe processed into various forms (e.g., crystalline Form 2, amorphous,etc.) as discussed further below. And the composition can be formulatedfor various routes of administration. In some embodiments, thecompositions can be used to treating or ameliorating hyperammonemia orhepatic encephalopathy.

Compositions of L-Ornithine Phenyl Acetate

Also disclosed herein are compositions of L-ornithine phenyl acetatethat may be formed by the process disclosed herein. The compositions ofthe present application advantageously have low amounts of inorganicsalts, particularly alkali metal salts and/or halide salts, andtherefore are particularly suited for oral and/or intravenousadministration to patients with hepatic encephalopathy. Meanwhile, thesecompositions may exhibit similar stability profiles compared to othersalts (e.g., mixtures of L-ornithine hydrochloride and sodium phenylacetate). The compositions may, in some embodiments, be obtained by oneof the processes disclosed in the present application. For example, anyof the disclosed processes using L-ornithine benzoate as an intermediatemay yield the compositions of the present application.

The compositions, in some embodiments, can include a crystalline form ofL-ornithine phenyl acetate (e.g., Forms I, II, III and/or V disclosedherein). In some embodiments, the composition may include at least about20% by weight of a crystalline form of L-ornithine phenyl acetate(preferably at least about 50% by weight, and more preferably at leastabout 80% by weight). In some embodiments, the composition consistsessentially of a crystalline form of L-ornithine phenyl acetate. In someembodiments, the composition includes a mixture of at least two (e.g.,two, three or four forms) of Forms I, II, III, and V.

The compositions, in some embodiments, include Form II. For example, thecompositions may include at least about 20%; at least about 50%; atleast about 90%; at least about 95%; or at least about 99% of Form II.Similarly, the compositions may also include, for example, Forms I, IIIor V. The compositions may optionally include at least about 20%; atleast about 50%; at least about 90%; at least about 95%; or at leastabout 99% of Forms I, II, III and/or V.

Also within the scope of the present application are amorphous forms ofL-ornithine phenyl acetate. Various methods are known in the art forpreparing amorphous forms. For example, a solution of L-ornithine phenylacetate may be dried under vacuum by lyophilization to obtain anamorphous composition. See P.C.T. Application WO 2007/058634, whichpublished in English and designates the U.S., and is hereby incorporatedby reference for disclosing methods of lyophilization.

It is preferred that the composition have low amounts (if any) of alkaliand halogen ions or salts, particular sodium and chloride. In someembodiments, the composition comprises no more than about 100 ppm ofalkali metals (preferably no more than about 20 ppm, and most preferablyno more than about 10 ppm). In some embodiments, the compositioncomprises no more than about 100 ppm of sodium (preferably no more thanabout 20 ppm, and most preferably no more than about 10 ppm). In someembodiments, the composition comprises no more than about 0.1% by weightof halides (preferably no more than about 0.01% by weight). In someembodiments, the composition comprises no more than about 0.1% by weightof chloride (preferably no more than about 0.01% by weight).

The reduced content of alkali metals and halides provides a compositionsuitable for preparing concentrated isotonic solutions. As such, thesecompositions can be more easily administered intravenously compared to,for example, administering mixtures of L-ornithine hydrochloride andsodium phenyl acetate. In some embodiments, an about 45 to about 55mg/mL solution of L-ornithine phenyl acetate in water (preferably about50 mg/mL) is isotonic with body fluids (e.g., the solution exhibits anosmolality in the range of about 280 to about 330 mOsm/kg).

The compositions may also include residual amounts of the anion from anintermediate salt formed during the process of making the L-ornithinephenyl acetate composition. For example, some of the processes disclosedherein yield compositions having benzoic acid or a salt thereof. In someembodiments, the composition comprises at least about 0.01% by weightbenzoic acid or a salt thereof (preferably at least about 0.05% byweight, and more preferably about 0.1% by weight). In some embodiments,the composition comprises no more than about 3% by weight benzoic acidor a salt thereof (preferably no more than about 1% by weight, and morepreferably no more than about 0.5% by weight). In some embodiments, thecomposition includes a salt, or an acid thereof, in the range of about0.01% to about 3% by weight (preferably about 0.1% to about 1%), whereinthe salt is selected from acetate, aspartate, formate, oxalate,bicarbonate, carbonate, sulfate, nitrate, isonicotinate, salicylate,citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzensulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate) or phosphate.

Similarly, a composition prepared using an acetate intermediate may haveresidual amounts of acetic acid or acetate. In some embodiments, thecomposition includes at least about 0.01% by weight acetic acid oracetate (preferably at least about 0.05% by weight, and more preferablyabout 0.1% by weight). In some embodiments, the composition includes nomore than about 3% by weight acetic acid or acetate (preferably no morethan about 1% by weight, and more preferably no more than about 0.5% byweight).

The compositions may also include low amounts of silver. Exemplaryprocesses disclosed herein utilize, for example, silver benzoate, butstill yield compositions with surprisingly low amounts of silver. Thus,in some embodiments, the composition includes no more than about 600 ppmsilver (preferably no more than about 100 ppm, and more preferably nomore than about 65 ppm). In some embodiments, the composition includesat least about 10 ppm silver (alternatively at least about 20 or 25 ppmsilver).

Pharmaceutical Compositions

The compositions of L-ornithine phenyl acetate prepared by the processesdisclosed above may also be formulated for administration to a subject(e.g., a human). L-Ornithine phenyl acetate, and accordingly thecompositions disclosed herein, may be formulated for administration witha pharmaceutically acceptable carrier or diluent. L-ornithine phenylacetate may thus be formulated as a medicament with a standardpharmaceutically acceptable carrier(s) and/or excipient(s) as is routinein the pharmaceutical art. The exact nature of the formulation willdepend upon several factors including the desired route ofadministration. Typically, L-ornithine phenyl acetate is formulated fororal, intravenous, intragastric, subcutaneous, intravascular orintraperitoneal administration.

The pharmaceutical carrier or diluent may be, for example, water or anisotonic solution, such as 5% dextrose in water or normal saline. Solidoral forms may contain, together with the active compound, diluents,e.g. lactose, dextrose, saccharose, cellulose, corn starch or potatostarch; lubricants, e.g. silica, talc, stearic acid, magnesium orcalcium stearate, and/or polyethylene glycols; binding agents, e.g.starches, gum arabic, gelatin, methylcellulose, carboxymethylcelluloseor polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginicacid, alginates or sodium starch glycolate; effervescing mixtures;dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates,laurylsulphates; and, in general, non-toxic and pharmacologicallyinactive substances used in pharmaceutical formulations. Suchpharmaceutical preparations may be manufactured in known manners, forexample, by means of mixing, granulating, tabletting, sugar-coating, orfilm-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions orsuspensions. The syrups may contain as carriers, for example, saccharoseor saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain a carrier, for example a naturalgum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol. The suspensions orsolutions for intramuscular injections may contain, together withL-ornithine phenyl acetate, a pharmaceutically acceptable carrier, e.g.sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol,and if desired, a suitable amount of lidocaine hydrochloride.

The medicament may consist essentially of L-ornithine phenyl acetate anda pharmaceutically acceptable carrier. Such a medicament thereforecontains substantially no other amino acids in addition to L-ornithineand phenyl acetate. Furthermore, such a medicament containsinsubstantial amounts of other salts in addition to L-ornithine phenylacetate.

Oral formulations may generally include dosages of L-ornithine phenylacetate in the range of about 500 mg to about 100 g. Accordingly, insome embodiments, the oral formulation includes the L-ornithine phenylacetate compositions disclosed herein in the range of about 500 mg toabout 50 g. In some embodiments, the oral formulation is substantiallyfree of alkali metal salts and halides (e.g., contains no more thantrace amounts of alkali metal salts and halides).

Intravenous formulations may also generally include dosages ofL-ornithine phenyl acetate in the range of about 500 mg to about 100 g(preferably about 1 g to about 50 g). In some embodiments, theintravenous formulation is substantially free of alkali metal salts andhalides (e.g., contains no more than trace amounts of alkali metal saltsand halides). In some embodiments, the intravenous formulation has aconcentration of about 5 to about 300 mg/mL of L-ornithine phenylacetate (preferably about 25 to about 200 mg/mL, and more preferablyabout 40 to about 60 mg/mL).

The composition, or medicament containing said composition, mayoptionally be placed is sealed packaging. The sealed packaging mayreduce or prevent moisture and/or ambient air from contacting thecomposition or medicament. In some embodiments, the packaging includes ahermetic seal. In some embodiments, the packaging sealed under vacuum orwith an inert gas (e.g., argon) within the sealed package. Accordingly,the packaging can inhibit or reduce the rate of degradation for thecomposition or medicament stored within the packaging. Various types ofsealed packaging are known in the art. For example, U.S. Pat. No.5,560,490, is hereby incorporate by reference in its entirety, disclosesan exemplary sealed package for medicaments.

Crystalline Forms of L-Ornithine Phenyl Acetate

Also disclosed herein are crystalline forms of L-ornithine phenylacetate, and in particular, crystalline Form I, Form II, Form III, andForm V. L-Ornithine phenyl acetate may, in some embodiments, be obtainedusing the processes disclosed above and then crystallized using any ofthe methods disclosed herein.

Form I

The precise conditions for forming crystalline Form I may be empiricallydetermined and it is only possible to give a number of methods whichhave been found to be suitable in practice.

Thus, for example, crystalline Form I may generally be obtained bycrystallizing L-ornithine phenyl acetate under controlled conditions. Asan example, precipitating L-ornithine phenyl acetate from a saturatedsolution by adding ethanol at reduced temperatures (e.g., 4° or −21°C.). Exemplary solvents for the solution that yield crystalline Form Iupon adding ethanol include, but are not limited to, cyclohexanone,1-propanol, diemthylcarbonate, N-methylpyrrolidine (NMP), diethyl ether,2-butanol, cumene, ethyl formate, isobutyl acetate, 3-methyl-1-butanol,and anisole.

Accordingly, in the context of the processes for making L-ornithinephenyl acetate disclosed above, the process can yield Form I byutilizing particular isolation methods. For example, L-ornithine phenylacetate may be isolated by adding ethanol at reduced temperature toyield Form I.

Crystalline Form I was characterized using various techniques which aredescribed in further detail in the experimental methods section. FIG. 1shows the crystalline structure of Form I as determined by X-ray powderdiffraction (XRPD). Form I, which may be obtained by the methodsdisclosed above, exhibits characteristic peaks at approximately 4.9°,13.2°, 17.4°, 20.8° and 24.4° 2θ. Thus, in some embodiments, acrystalline form of L-ornithine phenyl acetate has one or morecharacteristic peaks (e.g., one, two, three, four or five characteristicpeaks) selected from approximately 4.9°, 13.2°, 17.4°, 20.8°, and 24.4°2θ.

As is well understood in the art, because of the experimentalvariability when X-ray diffraction patterns are measured on differentinstruments, the peak positions are assumed to be equal if the two theta(2θ) values agree to within 0.2° (i.e., ±0.2°). For example, the UnitedStates Pharmacopeia states that if the angular setting of the 10strongest diffraction peaks agree to within ±0.2° with that of areference material, and the relative intensities of the peaks do notvary by more than 20%, the identity is confirmed. Accordingly, peakpositions within 0.2° of the positions recited herein are assumed to beidentical.

FIG. 2 shows results obtained by differential scanning calorimetry (DSC)for Form I. These results indicate an endotherm at 35° C., which ispossibly associated with a desolvation and/or dehydration to Form II. Asecond transition at about 203° C. indicates the melting point for thecrystal. To explore the possible existence of a desolvation and/ordehydration transition, Form I was analyzed by thermogravimetricgravimetric/differential thermal analysis (TG/DTA), which is shown inFIG. 3. Form I exhibits a 11.28% weight loss at about 35° C., andtherefore these results further suggest that Form I exhibits adesolvation and/or dehydration transition at about 35° C. The meltingpoint of about 203° C. could also be observed by TGA testing.Accordingly, in some embodiments, the crystalline form of L-ornithinephenyl acetate is characterized by differential scanning calorimetry ashaving an endotherm at about at about 35° C. In some embodiments, acrystalline form of L-ornithine phenyl acetate exhibits a weight loss ofabout 11% at about 35° C., as determined by TGA. In some embodiments, acrystalline form of L-ornithine phenyl acetate exhibits a melting pointof about 203° C.

FIG. 4 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form I. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH₂), 3.6 (CH₂unit of phenyl acetate), 3.15 (CH₂ adjacent to NH₂) and 1.9 (aliphaticCH₂ units) ppm (integrals: 5:1:2:2:4 protons; 1.2, 0.25, 0.5, 0.5, 1.0).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 5 shows dynamic vapor sorption (DVS) results for Form I, and show awater uptake of about 0.2% by weight. XRPD results following DVAanalysis (not shown) confirm that Form I did not transition to adifferent polymorph. Form I can therefore be characterized asnon-hygroscopic and stable over a wide range of humidity.

A 7-day stability study of Form I at 40° C./75% RH indicated that atransformation to Form II occurred under these conditions. Form I alsoconverts to Form II at elevated temperatures (e.g., 80° or 120° C.),with or without applying a vacuum, after 7 or 14 days. Accordingly, FormI is metastable.

Single crystal x-ray diffraction (SXRD) was also used to determine thestructure of Form I at −20° and −123° C., and the results are summarizedin TABLES 1 and 2. The results confirm that Form I is a solvate havingethanol and water molecules within the unit cell. In some embodiments, acrystalline form of L-ornithine phenyl acetate can be represented by theformula C₁₅H₂₈N₂O₆. In some embodiments, a crystalline form ofL-ornithine phenyl acetate can be represented by the formula[C₅H₁₃N₂O₂][C₈H₇O₂]EtOH.H₂O. In some embodiments, a crystalline form ofL-ornithine phenyl acetate exhibits a single crystal X-raycrystallographic analysis with crystal parameters approximately equal tothe following: unit cell dimensions of a=5.3652(4) Å, b=7.7136(6) Å,c=20.9602(18) Å, α=90°, β=94.986(6°), γ=90°; a monoclinic crystalsystem, and a P2₁ space group.

TABLE 1 Crystallographic Data of Form I Collected at −20° C. EmpiricalFormula C₁₅ H₂₈ N₂ O₆ or [C₅H₁₃N₂O₂][C₈H₇O₂]EtOH•H₂O Formula Weight332.39 Crystal System Monoclinic Space Group P2₁ Unit Cell Dimensions a= 5.3652(4) Å α = 90° b = 7.7136(6) Å β = 94.986(6)° c = 20.9602(18) Å γ= 90° Volume 864.16(12) Å³ Number of Reflections 1516 (2.5° < θ < 28°)Density (calculated) 1.277 mg/cm³

TABLE 2 Crystallographic Data of Form I Collected at −123° C. EmpiricalFormula C₁₅ H₂₈ N₂ O₆ or [C₅H₁₃N₂O₂][C₈H₇O₂]EtOH•H₂O Formula Weight332.39 Crystal System Monoclinic Space Group P2₁ Unit Cell Dimensions a= 5.3840(9) Å α = 90° b = 7.7460(12) Å β = 95.050(12)° c = 21.104(4) Å γ= 90° Volume 876.7(3) Å³ Number of Reflections 1477 (2.5° < θ < 18°)Density (calculated) 1.259 mg/cm³

Form II

The precise conditions for forming crystalline Form II may beempirically determined and it is only possible to give a number ofmethods which have been found to be suitable in practice.

Thus, for example, crystalline Form II may be prepared bycrystallization under controlled conditions. Crystalline Form II can beprepared by, for example, evaporating a saturated organic solution ofL-ornithine phenyl acetate. Non-limiting examples of organic solutionsthat may be used to obtain Form II include ethanol, acetone,benzonitrile, dichloromethane (DCM), dimethyl sulfoxide (DMSO), ethylacetate (EtOAc), acetonitrile (MeCN), methyl acetate (MeOAc),nitromethane, tert-butyl methyl ether (TBME), tetrahydrofuran, andtoluene. Other solvents may yield a mixture of Form I and II, such as,but not limited to, 1,4 dioxane, 1-butanol, cyclohexane, IPA, THF, MEK,MeOAc and water.

Form II can also be obtained by precipitating L-ornithine phenyl acetatefrom a saturated organic solution by adding an anti-solvent forL-ornithine phenyl acetate, such as IPA. Form II may be precipitatedover a broad range of temperatures (e.g., room temperature, 4° C., and−21° C.). Non-limiting examples of suitable solvents for the saturatedorganic solution include cyclohexanone, 1-propanol, dimethyl carbonate,N-methylpyrrolidone (NMP), diisopropyl ether, diethyl ether, ethyleneglycol, dimethylformamide (DMF), 2-butanol, cumene, isobutyl acetate,3-methyl-1-butanol, and anisole. Alternatively, the same listed solvents(e.g., cyclohexanone) can be used to form a solution of L-ornithinephenyl acetate, and Form II may be precipitated by adding ethanol atambient conditions. As another example, Form II may also be obtained byforming a slurry of L-ornithine phenyl acetate with the listed organicsolvents and cycling the temperature between 25° and 40° C. every 4hours for about 18 cycles (or 72 hours).

Accordingly, in the context of the processes for making L-ornithinephenyl acetate disclosed above, the process can yield Form II byutilizing particular isolation methods. For example, L-ornithine phenylacetate may by isolated by adding IPA, or evaporating the organicsolvent, to yield Form II.

FIG. 6 shows the crystalline structure of Form II as determined by XRPD.Form II, which may be obtained by the methods disclosed above, exhibitscharacteristic peaks at approximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8°and 24.1° 2θ. Thus, in some embodiments, a crystalline form ofL-ornithine phenyl acetate has one or more characteristic peaks (e.g.,one, two, three, four, five or six characteristic peaks) selected fromapproximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.12° θ.

FIG. 7 shows results obtained by differential scanning calorimetry (DSC)for Form II. These results indicate a melting point of about 202° C.,which is approximately the same as the melting point for Form I. Thissuggests that Form I transitions to Form II upon heating above about 35°C. Form II was also analyzed using TG/DTA, as shown in FIG. 8, andexhibits an about 9.7% weight loss associated with residual solvent. Themelting point of about 202° C. could also be observed by TGA testing.Accordingly, in some embodiments, a crystalline form of L-ornithinephenyl acetate exhibits a melting point of about 202° C.

A 7-day stability study of Form II at 40° C./75% RH failed to produce anobservable phase change. In fact, Form II was stable over 14 days whenexposed to elevated temperatures, varying pHs, UV light or oxygen.Accordingly, Form II is considered stable.

FIG. 9 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form II. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH2), 3.6 (CH2unit of phenylacetate), 3.15 (CH2 adjacent to NH2) and 1.9 (aliphaticCH2 units) ppm (integrals: 5:1:2:2:4 protons; 7.0, 1.4, 2.9, 3.0, 5.9).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 10 shows dynamic vapor sorption (DVS) results for Form II, and showa water uptake of about 0.3% by weight. XRPD results following DVAanalysis (not shown) confirm that Form II did not transition to adifferent polymorph. Form II can therefore be characterized asnon-hygroscopic and stable over a wide range of humidity.

Single crystal x-ray diffraction (SXRD) was also used to determine thestructure of Form II at 23° and −123° C., and the results are summarizedin TABLES 3 and 4. The results demonstrate that Form II is anhydrous andtherefore structurally different from Form I. In some embodiments, acrystalline form of L-ornithine phenyl acetate can be represented by theformula C₁₃H₂₀N₂O₄. In some embodiments, a crystalline form ofL-ornithine phenyl acetate can be represented by the formula[C₅H₁₃N₂O₂][C₈H₇O₂]. In some embodiments, a crystalline form ofL-ornithine phenyl acetate exhibits a single crystal X-raycrystallographic analysis with crystal parameters approximately equal tothe following: unit cell dimensions of a=6.594(2) Å, α=90°, b=6.5448(18)Å, β=91.12(3)°, c=31.632(8) Å, γ=90°; a monoclinic crystal system; and aP2₁ space group.

TABLE 3 Crystallographic Data of Form II Collected at 23° C. EmpiricalFormula C₁₃H₂₀N₂O₄ or [C₅H₁₃N₂O₂][C₈H₇O₂] Formula Weight 268.31 CrystalSystem Monoclinic Space Group P2₁ Unit Cell Dimensions a = 6.594(2) Å α= 90° b = 6.5448(18) Å β = 91.12(3)° c = 31.632(8) Å γ = 90° Volume1364.9(7) Å³ Number of Reflections 3890 (3° < θ < 20.5°) Density(calculated) 1.306 mg/cm³

TABLE 4 Crystallographic Data of Form II Collected at −123° C. EmpiricalFormula C₁₅ H₂₈ N₂ O₆ or [C₅H₁₃N₂O₂][C₈H₇O₂] Formula Weight 332.39Crystal System Monoclinic Space Group P2₁ Unit Cell Dimensions a =5.3652(4) Å α = 90° b = 7.7136(6) Å β = 94.986(6)° c = 20.9602(18) Å γ =90° Volume 864.16(12) Å³ Number of Reflections 1516 (2.5° < θ < 28°)Density (calculated) 1.277 mg/cm³

Form III

The precise conditions for forming crystalline Form III may beempirically determined and it is only possible to give a number ofmethods which have been found to be suitable in practice.

Thus, for example, Form III may be obtained by placing a saturatedsolution of L-ornithine phenyl acetate in a cooled temperatureenvironment of about −21° C., where the solution is a mixture of acetoneand water (e.g., equal parts volume of acetone and water). As anotherexample, adding IPA to a saturated solution of L-ornithine phenylacetate in 2-butanol can yield Form III when completed at ambientconditions. Furthermore, Form III may be obtained, for example, byadding IPA to a saturated solution of L-ornithine phenyl acetate inisobutyl acetate when completed at reduced temperatures of about −21° C.

Accordingly, in the context of the processes for making L-ornithinephenyl acetate disclosed above, the process can yield Form III byutilizing particular solvents and isolation methods. For example,L-ornithine phenyl acetate may be formed within a mixture of acetone andwater, and subsequently placed in a cool environment of about −21° C. toyield Form III.

FIG. 11 shows the crystalline structure of Form III as determined byXRPD. Form III, which may be obtained by the methods disclosed above,exhibits characteristic peaks at approximately 5.8°, 14.1°, 18.6°,19.4°, 22.3° and 24.8° 2θ. Thus, in some embodiments, a crystalline formof L-ornithine phenyl acetate has one or more characteristic peaks(e.g., one, two, three, four, five or six characteristic peaks) selectedfrom approximately 5.8°, 14.1°, 18.6°, 19.4°, 22.3° and 24.8° 2θ.

FIG. 12 shows results obtained by differential scanning calorimetry(DSC) for Form III. These results indicate a melting point of about 203°C., which is approximately the same as the melting points for Form I andForm II. Additionally, Form III exhibits an endotherm at about 40° C.Form III was also analyzed using TG/DTA, as shown in FIG. 13, andexhibits no significant weight loss before the melting point. Form IIImay therefore be characterized as anhydrous. The melting point of about203° C. could also be observed by TGA testing. Accordingly, in someembodiments, a crystalline form of L-ornithine phenyl acetate exhibits amelting point of about 203° C. In some embodiments, a crystalline formof L-ornithine phenyl acetate is characterized by differential scanningcalorimetry as having an endotherm at about 40° C. In some embodiments,a crystalline form of L-ornithine phenyl acetate is anhydrous.

A 7-day stability study of Form III at 40° C./75% RH indicated that atransformation to Form II occurred under these conditions. In contrast,Form II is stable at elevated temperatures, with or without vacuum, forperiods of 7 or 10 days. Accordingly, Form III is most likelymetastable, but more stable than Form I.

FIG. 14 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form III. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH2), 3.6 (CH2unit of phenyl acetate), 3.15 (CH2 adjacent to NH2) and 1.9 (aliphaticCH2 units) ppm (integrals: 5:1:2:2:4 protons; 4.2, 0.8, 1.7, 1.7, 3.0).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 15 shows dynamic vapor sorption (DVS) results for Form III, andshow a water uptake of about 2.0% by weight. XRPD results following DVSanalysis (not shown) confirm that Form III did not transition to adifferent polymorph. Form III therefore exhibits greater water uptakecompared to Forms I and II; however Form III is still characterized asnon-hygroscopic and stable over a wide range of humidity at roomtemperature.

Form V

The precise conditions for forming crystalline Form V may be empiricallydetermined and it is only possible to give a number of methods whichhave been found to be suitable in practice.

Thus, for example, Form V may be obtained by placing a saturatedsolution of L-ornithine phenyl acetate in a cooled temperatureenvironment of about −21° C., where the solution is cyclohexanone. Asanother example, the same saturated solution may yield Form V whenevaporating the solvent.

Form V also forms from saturated solutions of L-ornithine phenyl acetatehaving diisopropyl ether as a solvent. For example, a saturated solutionhaving a solvent ratio of about 1 to 2 of diisopropyl ether and IPA willyield Form V when placed in a cooled temperature environment of about 4°C. Similarly, a solution having only the solvent diisopropyl ether canyield Form V when placed in a cooled temperature environment of about−21° C.

FIG. 16 shows the crystalline structure of Form V as determined by XRPD.Form V, which may be obtained by the methods disclosed above, exhibitscharacteristic peaks at approximately 13.7°, 17.4°, 19.8°, 20.6° and23.7° 2θ. Thus, in some embodiments, a crystalline form of L-ornithinephenyl acetate has one or more characteristic peaks (e.g., one, two,three, four, or five characteristic peaks) selected from approximately13.7°, 17.4°, 19.8°, 20.6° and 23.7° 2θ.

FIG. 17 shows results obtained by differential scanning calorimetry(DSC) for Form V. These results indicate a melting point of about 196°C., which is below the melting point of other forms. Form V alsoexhibits an endotherm at about 174° C. Form V was also analyzed usingthermal gravimetric analysis (TGA), as shown in FIG. 18, and exhibits nosignificant weight loss before the melting point. Form V may thereforebe characterized as anhydrous. The melting point of about 196° C. couldalso be observed by TGA testing. Accordingly, in some embodiments, acrystalline form of L-ornithine phenyl acetate exhibits a melting pointof about 196° C. In some embodiments, a crystalline form of L-ornithinephenyl acetate is characterized by differential scanning calorimetry ashaving an endotherm at about 174° C. In some embodiments, a crystallineform of L-ornithine phenyl acetate is anhydrous.

FIG. 19 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form V. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH2), 3.6 (CH2unit of phenyl acetate), 3.15 (CH2 adjacent to NH2) and 1.9 (aliphaticCH2 units) ppm (integrals: 5:1:2:2:4 protons; 4.2, 0.8, 1.7, 1.7, 3.0).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 19 shows dynamic vapor sorption (DVS) results for Form V, and showa water uptake of about 0.75% by weight. XRPD results following DVSanalysis (not shown) suggest that Form V transitioned to Form II, butthe chemical composition was unchanged. Form V is thereforecharacterized as non-hygroscopic, but not stable over a wide range ofhumidity.

A 7-day stability study of Form V at 40° C./75% RH indicated that atransformation to Form II occurred under these conditions; however thechemical composition was unchanged. Accordingly, Form V is most likelymetastable.

Examples and Experimental Methods

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1 Small-Scale Batch Process to Produce L-Ornithine PhenylAcetate

About 8.4 g (0.049 moles) of L-ornithine hydrochloride was dissolved in42 mL H₂O and, separately, about 11.4 g of silver benzoate was dissolvedin 57 mL DMSO. Subsequently, the silver benzoate solution was added tothe L-ornithine hydrochloride solution. Combining the two mixturesresulted in an immediate, exothermic precipitation of a creamy whitesolid (AgCl). The solid was removed by vacuum filtration and retainingthe filtrate (L-ornithine benzoate in solution). 200 mL of IPA was addedto the filtrate and the mixture was cooled to 4° C. A crystalline solidprecipitated after about 3 hours (L-ornithine benzoate) which wasisolated by vacuum filtration. Yield: 60%.

7.6 g (0.03 moles) of the L-ornithine benzoate was dissolved in 38 mLH₂O and about 4.4 g of sodium phenyl acetate was dissolved 22 mL H₂O.Subsequently, the sodium phenyl acetate solution was added to theL-ornithine benzoate solution and left to stir for about 10 minutes.About 240 mL of IPA (8:2 IPA:H₂O) was added and the solution stirred for30 minutes before cooling to 4° C. A crystalline solid precipitatedafter about 3 hours at 4° C. (L-ornithine phenyl acetate). Theprecipitate was isolated by vacuum filtration and washed with 48-144 mLof IPA. Yield: 57%.

Example 2 Large-Scale Batch Process to Produce L-Ornithine PhenylAcetate

Two separate batches of L-ornithine phenyl acetate were prepared asfollows:

About 75 kg of L-Ornithine monohydrochloride was dissolved in 227 kg ofwater. To the resulting solution was added 102 kg of silver benzoatedissolved in 266 kg of DMSO at room temperature within 2 hours.Initially, a strong exothermy was observed and the silver chlorideprecipitated. The receiver containing the solution was then washed with14 kg of DMSO that was added to the reaction mass. In order to removethe silver chloride formed, the reaction mass was filtered over a lensfilter prepared with 10 kg of Celite and a GAF filter of 1 mm. Afterfiltration, the filter was washed with an additional 75 kg of water. Thereaction mass was placed in a different tank after filtration to preventcontamination with residual silver chloride. The reaction mass was thenheated at 35±2° C. and 80 kg of sodium phenyl acetate was added. At thispoint the reaction mass was stirred at 35±2° C. for at least 30 minutes.

In order to precipitate the final product, 353 kg of IPA was added tothe reaction mass. The reaction mass was then cooled to 0±3° C. within 6hours, stirred for 1 hour and then the product isolated in a centrifuge.

About 86 kg of final wet product was obtained. The product was thendried at 40±5° C. for about 6.5 to 8 hours to provide about 75 kg ofL-ornithine phenyl acetate. Yield: 63.25%. TABLE 5 summarizesmeasurements relating to the final product.

TABLE 5 Analytical Results for Large-scale Batch Process Test Batch 1Batch 2 Purity 98.80% 98.74% Benzoate  0.17%  0.14% Silver 28 ppm 157ppm Chloride 0.006% 0.005% Sodium  7 ppm  26 ppm Total Impurities  0.17% 0.14% Physical Form Form II Form II

Example 3 Preparing Sodium Phenyl Acetate Solution In Situ

Phenyl acetic acid (PAA) was dissolved in a solution of isopropanol.About 1 molar equivalent of sodium hydroxide was added to the solutionand stirred. The obtained solution was added dropwise to a solutionhaving about 1 molar equivalent of L-ornithine benzoate. L-ornithinephenyl acetate was precipitated from this solution using generally thesame procedures described in Example 2. Yield: 53.5%. The white powderwas further characterized and is summarized in TABLE 6 under the heading“Trial A.”

Example 4 Water/IPA Solvent Ratios for Isolating L-Ornithine PhenylAcetate

Several studies were completed to consider the effect of the solventcomposition on the yield and product purity. The trials were completedusing a similar protocol to Examples 2 and 3 and detailed in TABLE 6.

Trials A, B, and D include various ratios of water/IPA and demonstratethat yield can be improved by increasing the relative amount of IPA.Trial D crystallized the product upon intermixing the IPA, while TrialsA and B include cooling the solution to obtain the product. In addition,Trial F demonstrates that reducing the volume of reaction mass may alsofacilitate precipitation of the final product.

Trials C, E, and G include a distillation step to remove water from thereaction mass prior to adding IPA. The yield for Trials C, E, and G was70.0%, 51.2%, and 68.0%, respectively.

TABLE 6 Experimental Results for Water/IPA ratio Trial Water/IPAPrecipitation Product Description ratio Conditions Yield (%) DescriptionTRIAL A solvent ratio for 41:59 during cooling 53.5% white final productto 0° C. powder TRIAL B precipitation similar 46:54 50.5% white toExamples 2, 3 powder TRIAL C distillation introduced Unknown during70.0% off-white prior to IPA dosage distillation powder TRIAL Dincreased IPA ratio 30:70 during IPA 61.2% white to improve yield dosagepowder TRIAL E distillation introduced Unknown during IPA 51.2% whiteprior to IPA addition dosage powder to concentrate mass TRIAL Fdecreased water/IPA 46:54 during IPA 51.5% white volume to facilitatedosage powder product precipitation TRIAL G decreased water ratio and35:65 during PAA 68.0% white introduced distillation dosage powder afterfiltering

Example 5 Reducing Silver Content in L-Ornithine Phenyl Acetate

Batch 2 from Example 2 exhibited higher amounts of silver (157 ppm), andtherefore procedures were tested for reducing the silver content. Ninetrials were completed; each generally including dissolving about 20 g ofL-ornithine phenyl acetate from Batch 2 into 1.9 parts water, and thensubsequently adding 10.8 parts IPA. A crystalline form was isolated at0° C. by filtration.

For four trials, 8.0 mg or 80 mg of heavy metal scavengers SMOPEX 102 orSMOPEX 112 were added to the aqueous solution and stirred for 2 hours.The scavengers failed to reduce the silver content below 126 ppm. Instill another trial, the L-ornithine phenyl acetate was crashed out in asolution of IPA, rather than crystallized; however this trial alsofailed to reduce the silver content below 144 ppm.

The last three trials included adding diluted HCl to the solution toprecipitate remaining amount of silver as AgCl. The precipitate was thenremoved by filtration. The three trials included adding: (1) 1.0 g of0.33% HCl at 20° C.; (2) 1.0 g of 0.33% HCl at 30° C.; and (3) 0.1 g of3.3% HCl at 20° C. The three trials reduced the silver content to 30ppm, 42 ppm, and 33 ppm, respectively, and each trial yielding greaterthan 90% L-ornithine phenyl acetate. Accordingly, the addition of HClwas effective in reducing the amount of residual silver.

Example 6 Process for Preparing L-Ornithine Phenyl Acetate fromL-Ornithine Free Base by Using an Alkali Metal Alkoxide Base

As a general procedure, L-ornithine hydrochloride was suspended in asolvent. Subsequently, the reaction mass was heated and a base, sodiummethoxide, was added. NaCl formed and was removed from the system byfiltration. The reaction mass was cooled and a molar equivalent ofphenyl acetic acid was added to the reaction mass to form L-ornithinephenyl acetate. The final product was isolated, washed and dried. Asummary of the trials for this process is provided in TABLE 7.

TABLE 7 Process Trials Trial Base Eq. of Base Solvent 1 NaOMe 21% inMeOH 1.0 eq. MeOH 2 NaOMe 21% in MeOH 0.95 eq.  IPA 3 NaOMe 21% in EtOH1.0 eq. EtOH 4 NaOMe 21% in MeOH 1.0 eq. MeOH 5 NaOMe 21% in MeOH 1.0eq. MeOH w/IPA for precipitation 6 NaOMe 21% in MeOH 1.0 eq.Acetonitrile 7 NaOMe 21% in MeOH 1.0 eq. Water/IPA 8 NaOMe 21% in MeOH1.0 eq. Water/IPA 9 NaOMe 21% in MeOH 1.0 eq. n-butanol

The resulting L-ornithine phenyl acetate was found to exhibit highamounts of chloride (at least about 1% by weight), and presumablyinclude similar amounts of sodium. The yields were about 50% for Trials2, 4, and 5.

Example 7 Process for Preparing L-Ornithine Phenyl Acetate without anIntermediate Salt

Further studies were completed using generally the same procedure asExample 6. The results are shown in TABLE 8:

TABLE 8 Additional Process Trials Trial I Trial II Trial III Trial IVTrial 1 eq of 1 eq of 1 eq of 0.5 eq of description MeONa in MeONa inMeONa in Ca(OH)₂ in 10.6 p of 10.6 p of 15 p of 10.6 p of MeOH i-PrOHi-PrOH EtOH Yield 47.2% 41.9% 57.6% 40.2% Description white whiteoff-white white powder powder powder powder

Example 8 Process for Preparing L-Ornithine Phenyl Acetate fromL-Ornithine Free Base by Using a Carbonate Base

1 part L-ornithine hydrochloride by mole was suspended in about 10.6parts ethanol. Subsequently, the reaction mass (suspension) was heatedto about 50° C. and about 1 part calcium carbonate was added. Thereaction mass was stirred for about 2 hours. The free L-ornithine basewas isolated by filtration to obtain a powder, while the CaCl₂ remainedin solution. The filtered powder was dissolved in water and filtered toremove unreacted calcium carbonate. About 1 part phenyl acetic acid bymole in isopropanol was intermixed with the aqueous L-ornithinesolution. The final product precipitated from solution and was isolated,washed, and dried. Yield: 44.5%.

Example 9 Process for Preparing L-Ornithine Phenyl Acetate fromL-Ornithine Free Base by Using an Inorganic Base

1 part L-ornithine hydrochloride by mole was suspended in about 10.6parts ethanol. Subsequently, the reaction mass (suspension) was heatedto about 50° C. and about 0.5 part calcium hydroxide by mole was added.The reaction mass was stirred for about 1.5 hours. The free L-ornithinebase was isolated by filtration and washed with ethanol. The filteredsolid was dissolved in water and phenyl acetic acid (1.0 equivalent) inisopropanol was added dropwise to the aqueous L-ornithine solution atroom temperature. After at least 30 minutes stirring at roomtemperature, IPA was added to precipitate the final product. The finalproduct was isolated, washed, and dried. Yield: 43.95%.

Alternatively, L-ornithine free base was also prepared in an aqueoussolution. 1 part L-ornithine hydrochloride by mole was dissolved inabout 4.1 parts water. Subsequently, about 0.5 part calcium hydroxide bymole was added to the solution and stirred for about 30 minutes. Thefree L-ornithine base was then not isolated by filtration. Next, about 1part phenyl acetic acid in isopropanol was intermixed with the aqueousL-ornithine solution. The final product precipitated from solution wasisolated, washed, and dried. Yield: >100%.

Example 10 Process for Preparing L-Ornithine Phenyl Acetate fromL-Ornithine Free Base by Using Barium Hydroxide

To 1.0 equivalent of L-ornithine hydrochloride in an aqueous solutionwas added 2.7 molar equivalent of barium hydroxide. Subsequently, thereaction mass was refluxed for 2 hours and then cooled to roomtemperature. Sulfuric acid (6N) was added slowly to acidify theresulting reaction mass until pH was about 1.5. The insoluble bariumsulfate formed was filtered through by using a 0.2 μm filter. Thefiltrate was then concentrated by distillation and neutralized to pH7-7.5 by adding a barium hydroxide solution. The barium sulfate saltformed was removed again. Finally, a solution of phenyl acetic acid(1.13 equivalents) with sodium hydroxide in IPA was added at 35° C. tothe resulting solution at 35° C. IPA was added and the reaction mixturewas cooled to 0° C. to in order to precipitate the final product. Thefinal product was isolated by filtration, washed with a mixture of waterand IPA, and dried. Yield: 37.2%.

Example 11 Process for Preparing L-Ornithine Phenyl Acetate Via anAcetate Intermediate

Dissolve 25 mg of L-ornithine hydrochloride in 5 vols of H₂O, and thenadd excess acetic acid (about 5 vols) to form a slurry. Subject theslurry to temperature cycling between 25° C. and 40° C. every 4 hoursfor about 3 days. Add 1 equivalent of phenyl acetic acid (with respectto L-ornithine) and stir for about 4-6 hours (possibly heat). Use IPA asan anti-solvent, add enough to obtain a ratio of 70:30 (IPA:H₂O).Isolate by vacuum filtration and dry for about 4-8 hours at 80° C. toremove any residual acetic acid.

Example 12 Process for Preparing L-Ornithine Phenyl Acetate from HalideL-Ornithine Salt and Phenyl Acetate Salt

L-ornithine monohydrochloride is dissolved in water at a concentrationof 300-350 g/Kg (˜3 volumes water). To the resulting solution is added 1molar equivalent of silver phenyl acetate in 2.5 volumes DMSO (0.4 g/g)at room temperature within 2 hours. The receiver containing the solutionis then washed with DMSO and added to the reaction mass. The silverchloride formed can be filtered through the reaction mass over a lensfilter prepared with Celite and a GAF filter. After filtration, thefilter is washed with an additional volume of water.

In order to precipitate the final product, IPA is added to the reactionmass to a final concentration range of 65-95% IPA. The reaction mass isthen cooled to 0±3° C. within 6 hours, stirred for 1 hour and then theproduct isolated in a centrifuge.

The isolated wet product is redisolved in an aqueous dilute HCl solution(0.33%) representing between 1-3.5 molar equivalents of HCl. Thereaction mass is filtered over a lens filter prepared with Celite and aGAF filter to remove the silver chloride formed. After filtration, thefilter is washed with an additional volume of water.

To precipitate the final product, IPA is added again to the reactionmass to a final concentration range of 60-80% IPA. The reaction mass isthen cooled to 0±3° C. within 6 hours, stirred for 1 hour and then theproduct is isolated in a centrifuge. The final product is then dried at40±5° C. for about 6.5 to 8 hours to provide crystalline L-ornithinephenyl acetate.

What is claimed is:
 1. A process for making L-ornithine phenyl acetatesalt comprising: preparing a solution of sodium phenyl acetate by mixinga phenyl acetic acid and sodium hydroxide in a solvent; intermixingL-ornithine benzoate with the solution of sodium phenyl acetate; andisolating a composition comprising L-ornithine phenyl acetate.